Method of extracting antigens of bacteria

ABSTRACT

A means for identifying C. jejuni, from stool specimens, by the extraction and agglutination of its soluble antigen(s). More specifically, bacteria are concentrated from fresh (unpreserved) stool specimens, soluble antigen is released--preferably either by a specified heating protocol or by the action of a particular enzyme--and the antigen is detected by agglutination upon exposure to monoclonal, polyclonal or other corresponding antibodies. The method of the invention not only fosters efficiency in the laboratory but also provides rapid diagnosis of the presence of C. jejuni, so that appropriate patient treatment may begin at the earliest opportunity.

FIELD OF THE INVENTION

The present invention relates to the identification, by their enzyme- orheat-soluble antigens, of pathogenic bacteria in feces, which bacteriaare causal of enteritis and related diseases in humans.

INTRODUCTION

Effective treatment of nonviral diarrheal illness (enteritis, dysentary,etc.) ordinarily requires accurate identification of the causalbacterial pathogen. Although the best known enteritis-causing bacteriainclude the Salmonella and Shigella species, the role of Campylobacterjejuni (C. jejuni) in human enteritis has recently been recognized aswell. Indeed, the organism has been found in 3 to 11 percent of patientswith diarrheal illness in Europe and North America. Moreover, it hasbeen isolated in virtually every country, whether temperate or tropical,in which it has been sought. Fecal specimens from patients with diarrheashould therefore be examined routinely for the presence of C. jejuni, inaddition to the other diagnostic tests which are already routinelyperformed.

C. jejuni is microaerophilic and requires a special environment forsuccessful incubation. The routine methods of stool culturing for thisreason ordinarily fail to isolate C. jejuni because, under conditionsadverse to its growth, the organism is quickly overgrown by thepredominant enteric flora. Laboratory identification of C. jejuni hastherefore ordinarily required separate culturing with special culturemedia and elevated temperatures, along with a total incubation time of48 to 72 hours.

As a result, in order to effect rapid diagnosis and treatment, a needremains for a method that will identify C. jejuni from stool specimensin a relatively short period of time, without culturing, and with aminimum of attention by the laboratory or other health care personnel.

BRIEF DESCRIPTION OF THE INVENTION

In order to meet this need, the present invention is a means foridentifying C. jejuni, from stool specimens, by the extraction andagglutination of its soluble antigen(s). More specifically, bacteria areconcentrated from stool specimens, soluble antigen isreleased--preferably either by a specified heating protocol or by theaction of a particular enzyme--and the antigen is detected byagglutination upon exposure to monoclonal, polyclonal or othercorresponding antibodies. The method of the invention not only fostersefficiency in the laboratory but also provides rapid diagnosis of thepresence of C. jejuni without culturing, so that appropriate patienttreatment may begin at the earliest opportunity.

DETAILED DESCRIPTION OF THE INVENTION

Recent investigations have been pursued on the serotypes ofCampylobacter specimens with autoclaved, boiled or formalized antigenpreparations. There have been no investigations to date, however, whichreport the use of soluble antigenic extracts for the detection of theantigens of C. jejuni, from either pure culture or from mixed culture ofbacteria associated with this organism. Accordingly, the presentinvention provides a direct identification technique for use withextracted bacteria found in fecal specimens.

The present method is therefore a simple diagnostic test, which may beperformed on fecal specimens (usually human fecal specimens), in orderto confirm or negate a suspected presence of C. jejuni therein.Confirmation is achieved by the agglutination of a soluble antigen by alatex agglutination or coagglutination test as well as otheragglutination tests. As described in detail below, the test may becompleted within 30 minutes and includes the three main phases ofconcentration, extraction, and agglutination.

CONCENTRATION

Concentration of C. jejuni from a stool specimen commences withhomogenization of an aliquot of the specimen. Watery stool specimensordinarily need little or no agitation to form a homogeneous suspension;for example, a tube or vial containing the aliquot need only be sharplysnapped with the fingers three or four times. If the stool is formed, afew ml. of physiological saline or buffer or a bacteriological broth(all well known in the art) is mixed into the stool sample in order toyield an emulsified-liquid aliquot. In either case, the suspension needhave no specific concentration or viscosity but need have only theappearance of a roughly homogeneous aqueous suspension.

After the feces homogenizate is prepared, the C. jejuni in the aliquotmay be concentrated by the preferred technique of sucrose gradientdifferential centrifugation. To carry out this preferred technique, 3-5mls. of 50 percent aqueous sucrose solution is pipetted into acentrifuge tube, and about 1-2 mls. of 25 percent aqueous sucrosesolution is then pipetted on top of the 50 percent sucrose solution. A 1ml. (approximately) aliquot of the fecal suspension is then pipettedatop the 25 percent sucrose solution. The tube is then centrifuged in atabletop centrifuge at about 3000 RPM for 15 minutes at roomtemperature. A band of bacteria, which includes any C. jejuni presentalong with other fecal flora, will result at the interface of the twosucrose solutions. The band of bacteria is removed with a Pasteurpipette or other appropriate pipette and is subjected to one of theextraction technique outlined in the next section.

An alternate technique for bacterial concentration is the technique ofPercoll gradient different centrifugation. Percoll (Pharmacia FineChemicals, Uppsala, Sweden) is a differential centrifugation mediumwhich has a density of 1.131 g/ml. 3-5 ml. of 50 percent aqueous Percollis added to a centrifuge tube, and approximately 1 ml. of fecessuspension is pipetted atop the Percoll. The tube is then centrifuged ina tabletop centrifuge at 200×g at room temperature for about 15 minutes.The bacterial layer which contains the C. jejuni sediments within thecenter of the Percoll solution at a specific density of 1.19 g/ml. Somefine fecal particulates may also be present in the bacterial layer, butmost of the nonbacterial fecal matter precipitates to the bottom of thecentrifuge tube.

It is conceivable that one skilled in the art might identify otherconcentration techniques, for C. jejuni, with a minimum ofexperimentation. Unfortunately, applicant has already established that a10-100 fold loss of organisms occurs with other concentration techniquessuch as Millipore filtration, etc. The present techniques describedabove, however, provide bacterial bands which maximize concentration ofC. jejuni while minimizing the presence of unwanted fecal material inthe concentrate. This maximized concentration of C. jejuni in turncontributes to maximized reliability in the results of the presentmethod; the greater the C. jejuni concentration, the more likely apositive identification of the species will be.

EXTRACTION

Once concentrated, the presence of C. jejuni in a band of fecal bacteriamay be confirmed by the extraction and identification of one or moresoluble antigens. One of the two preferred extraction methods is theheat extraction method which follows. About 0.5 to 1 ml. of the bacteriaband (derived in the previous section) is placed in a glass vial such asa centrifuge or test tube or other glass vial. The bacterial suspensionis heated to 60° to 80° C. in a hot block, incubator or other heatingunit adapted to receive a test vial and to hold it withoutcontamination. Heating time is ordinarily 5 minutes or less. Antigenextraction is complete once the bacterial band reaches the 60° to 80° C.temperature, although the bacterial specimen may subsequently be allowedto cool if desired.

The second preferred extraction method is the subtilopeptidaseextraction method. Subtilopeptidase, or Bacillus subtilis alkalineproteinase, is acquired or prepared by means known in the art. As withthe heat extraction technique, about 0.5 to 1 ml. of the bacteria band(of the previous section) is placed in a glass vial. Instead of heatingthe vial, however, approximately 1 to 4 ml. of a 0.1 mg./ml. aqueoussolution of subtilopeptidase is added to the vial. The vial is leftundisturbed for approximately 5 to 10 minutes, after which extraction iscomplete.

Applicant believes that the above two extraction techniques provide formaximum antigen extraction, and thus maximum sensitivity and reliabilityin the text method of the present invention. As a result, although otherextractions such as with comparable amounts of the enzymes trypsin,papain and pepsin appear to demonstrate effectiveness in most or allextractions, the above-described methods are preferred.

AGGLUTINATION

C. jejuni antisera is prepared as follows. Confluent bacterial growth onfour Campy-blood agar plates (Remel Labs, Lenexa, Kans.) is obtainedafter 48 hours at 42° C. in a gas generator chamber "Bio Bag," Type CFJ,Marion Scientific, Kansas City, Mo. The bacteria are transferred by wayof a flame-sterilized wire loop to 3 ml. of saline (0.85 percent NaCl).Six rabbits (preferably white New Zealand males) are then immunizedintramuscularly with formalin-treated Campylobacter suspensions incomplete Freund adjuvant. These animals are boosted one month later witha series of intravenous injections of the same bacterial suspensions insaline. Blood is obtained by cardiac puncture or via ear vein 1-10 daysafter the last injection and is allowed to clot. Sera is then collectedand stored at -20° C.

Alternately, a monoclonal or polyclonal antibody of C. jejuni may beprepared by heating a C. jejuni suspension in phosphate buffer to 80° C.for 15 minutes. The filtered solution (0.45 μ filter) is used as thebacterial vaccine for the induction of a polyclonal or monoclonalantibody preparation by means known in the art.

A drop of the antigen-extracted bacterial suspension is placed on aglass microscope slide. A drop of the antisera described above (or,generally, an agglutination or coagglutination reagent prepared withpolyclonal, monoclonal or other C. jejuni-specific antibody) is mixedwith the drop of bacterial suspension by, for example, deposition of adrop of antisera onto the drop of antigen-extracted bacterial suspensionand subsequently rocking the slide. The presence of extracted C. jejuniis indicated by an agglutination reaction that occurs within thefollowing two minutes. The reaction may be observed, with the naked eye,by transillumination of the slide. Microscope observation or dot-ELISAagglutination detection may be used if the necessary equipment isreadily available, but one of the advantages of the present invention isthat the simple method may be accomplished quickly with very basiclaboratory equipment i.e., vials, pipettes, glass slides and a tabletopcentrifuge. Ambient light will in many cases be sufficient fortransillumination of the slide.

If use of the dot-ELISA is desired, however, the following protocol maybe considered exemplary. Extracted bacterial aliquots are "dotted" ontonitrocellulose membranes, and the dots are allowed to dry. The membranesare then blocked with 75 microliter of 10 percent bovine serum albuminfor 1 hour. After the blocking solution is decanted, 50 percent of C.jejuni antisera (diluted in 1 percent gelatin in phosphate bufferedsaline) is added to each dot. After an hour's incubation at roomtemperature, the membrane is rinsed with water and washed three timeswith phosphate buffered saline. Consecutive additions of antisera may becompleted in the same fashion if desired. Coloration and examinationthen proceed by means known in the art.

One embodiment of the invention may be conducted in accordance with thefollowing examples.

EXAMPLE 1

Six New Zealand white male rabbits were immunized intramuscularly withformalin-treated Campylobacter suspensions in complete Freund adjuvant.A series of 3 booster inoculations was given one month later over aperiod of three days. Five days after the last injection, blood wasdrawn from ear veins in each rabbit. No attempt was made atexsanguination and no rabbit was sacrificed; all animals recovered. Seraseparated from clotted blood was stored at -20° C.

EXAMPLE 2

A conical base centrifuge tube was filled with four ml. of 50% percentaqueous sucrose solution. One ml. of 25 percent aqueous sucrose solutionwas then placed atop the 50% sucrose solution and a 1 ml. aliquot ofhomogenized watery stool specimen (from a patient with symptomaticenteritis) was added atop the 25 percent sucrose solution. The tube wascentrifuged in a tabletop centrifuge at about 3000 RPM for 15 minutes atroom temperature.

A C. jejuni-containing bacterial layer appeared at the interface of thetwo sucrose solutions. One ml. of the bacterial band was removed with apipette and was transferred to a glass test tube. The bacterial aliquotwas heated to 70° C. over a 4 minute period in a laboratory hot block.One drop of the heated bacterial aliquot was placed on a glassmicroscope slide. A drop of the antisera prepared in Example 1 wasdropped atop the bacterial drop, and the slide was rocked. A visibleagglutination reaction, which occurred one minute after the two dropswere mixed, confirmed the presence of C. jejuni in the fecal specimen.

Having described presently preferred embodiments of the invention, it isto be understood that they may be otherwise embodied within the scope ofthe appended claims. For example, although the primary contemplated useof the present method is in the extraction and detection of antigens ofC. jejuni, over time as the bacterial populations mutate and evolve thepresent method will undoubtedly have equal applicability to theidentification of other Campylobacter species which have similar solubleantigens.

I claim:
 1. A method of identifying the presence of Campylobacterspecies in fecal specimens, comprising:(a) concentrating Campylobacterorganisms from fecal specimens using a concentrating media having atleast two layers of different densities to segregate the Campylobacterorganisms between the layers; (b) extracting soluble antigens from saidCampylobacter organisms; and (c) reacting said antigens withCampylobacter antisera to yield a detectable agglutinate.
 2. The methodaccording to claim 1, wherein the concentrating media comprise sucrosegradient media, wherein said sucrose gradient media contains at leasttwo layers in which the sucrose concentrations differ.
 3. The methodaccording to claim 1, wherein the concentrating media comprise sucrosegradient medium having two layers, one layer comprising a 50% aqueoussucrose solution beneath a 25% aqueous sucrose solution; and, theextracting of solution antigens from said Campylobacter organisms isaccomplished by heating said Campylobacter organisms.
 4. The methodaccording to claim 1, wherein the extracting of soluble antigens fromsaid Campylobacter organisms is accomplished by heating saidCampylobacter organisms to between 60° and 80° C.
 5. The methodaccording to claim 1, wherein the extracting of soluble antigens fromsaid Campylobacter organisms is accomplished with an enzyme.
 6. Themethod according to claim 1, wherein the extracting of soluble antigensfrom said Campylobacter organisms is accomplished with subtilopeptidase.7. The method according to claim 1, wherein the reacting of saidantigens and Campylobacter antisera is on a microscope slide to yield avisible agglutinate.
 8. The method according to claim 7, wherein thereacting of said antigens and Campylobacter antisera is on a microscopeslide with rocking of said slide to yield a visible agglutinate.
 9. Themethod according to claim 8, wherein the reacting in step (c) yields avisible agglutinate within two minutes.